Technical issues in using robots to reproduce joint specific gait.

Reproduction of the in vivo motions of joints has become possible with improvements in robot technology and in vivo measuring techniques. A motion analysis system has been used to measure the motions of the tibia and femur of the ovine stifle joint during normal gait. These in vivo motions are then reproduced with a parallel robot. To ensure that the motion of the joint is accurately reproduced and that the resulting data are reliable, the testing frame, the data acquisition system, and the effects of limitations of the testing platform need to be considered. Of the latter, the stiffness of the robot and the ability of the control system to process sequential points on the path of motion in a timely fashion for repeatable path accuracy are of particular importance. Use of the system developed will lead to a better understanding of the mechanical environment of joints and ligaments in vivo.

Strength of medial structures of the knee joint are decreased by isolated injury to the medial collateral ligament and subsequent joint immobilization.

Past studies of the healing of the medial collateral ligament (MCL) in animal models have been conducted over a variety of healing intervals, some as early as 1 week. One concern with testing at early healing intervals is the difficulty in identifying and isolating the tissues that carry load. The purpose of this study was to determine if isolation of the MCL and healing time are critical factors in the assessment of structural strength in this model. Furthermore, the effect of immobilization on these critical factors was investigated. Our approach was to calculate the load-sharing ratio between the MCL and the MCL plus capsule. A 4 mm gap was created in the midsubstance of both hindlimb MCLs of 52 female New Zealand White rabbits (n=104). Of these, 29 rabbits had their right hindlimb pin immobilized (immobilized group), leaving the left hindlimb non-immobilized. Testing was performed at 3 (n=12), 6 (n=22), and 14 (n=24) weeks. The remaining 23 rabbits, which had both limbs non-immobilized (non-immobilized group), were tested at 3 (n=10), 6 (n=12), 14 (n=12), and 40 (n=12) weeks. For both groups, half of the specimens at each healing interval were used to test the MCL alone and half to test the MCL plus capsule, except for 3 week immobilized joints where only the MCL plus capsule was tested. Additionally, MCL (n=12), MCL plus capsule (n=6), and capsule alone (n=5) were tested from normal animals. The load-sharing ratio at MCL failure for the normal joint was 89%, suggesting an MCL-dominated response. For the non-immobilized group, the load-sharing ratio was 24% at 3 weeks of healing, suggesting a capsule-dominated response. At and after 6 weeks of healing, an MCL-dominated response was observed, with the ratio being 68% or greater. Thus, at less than 6 weeks of healing, the structural strength capabilities of the joint may be better represented by the medial structures rather than the isolated MCL. Immobilization delayed the transition from a capsule-dominated response to an MCL-dominated response in this model.

Efficient transfer of intact oligonucleotides into the nucleus of ligament scar fibroblasts by HVJ-cationic liposomes is correlated with effective antisense gene inhibition.

The efficacy of two different cationic liposomes, Lipofectin and hemagglutinating virus of Japan (HVJ)-cationic liposomes, on nuclear uptake of fluorescence-labeled phosphorothioate oligodeoxyribonucleotide (S-ODN) by ligament scar fibroblasts and suppression of decorin mRNA expression when antisense decorin S-ODN was transferred was investigated. There was no significant difference in nuclear uptake of fluorescent ODN between the two methods. However, only HVJ-cationic liposomes had a significant effect on suppression of decorin mRNA expression levels. To address the discrepancy, the molecular integrity of the transferred ODN in the cells was assessed by analysis of fluorescence resonance energy transfer (FRET) within double-fluorescence-labeled S-ODN. More than 70% of the ODN transfected by HVJ-cationic liposomes remained intact within the nucleus at 20 h after transfection, while the majority of the ODN transferred by Lipofectin was degraded at this point. These results suggest a strong relationship between the nuclear integrity of transfected antisense ODN and its suppression of target mRNA expression.

Compressive compared with tensile loading of medial collateral ligament scar in vitro uniquely influences mRNA levels for aggrecan, collagen type II, and collagenase.

To test the hypothesis that loading conditions can be used to engineer early ligament scar behaviors, we used an in vitro system to examine the effect that cyclic hydrostatic compression and cyclic tension applied to 6-week rabbit medial collateral ligament scars had on mRNA levels for matrix molecules, collagenase, and the proto-oncogenes c-fos and c-jun. Our specific hypothesis was that tensile stress would promote more normal mRNA expression in ligament whereas compression would lead to higher levels of mRNA for cartilage-like molecules. Femur (injured medial collateral ligament)-tibia complexes were subjected to a hydrostatic pressure of 1 MPa or a tensile stress of 1 MPa of 0.5 Hz for 1 minute followed by 14 minutes of rest. On the basis of a preliminary optimization experiment, this 15-minute testing cycle was repeated for 4 hours. Semiquantitative reverse transcription-polymerase chain reaction analysis was performed for mechanically treated medial collateral ligament scars with use of rabbit specific primer sets for types I, II, and III collagen, decorin, biglycan, fibromodulin, versican, aggrecan, collagenase, c-fos, c-jun, and a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase. Cyclic hydrostatic compression resulted in a statistically significant increase in mRNA levels of type-II collagen (171% of nonloaded values) and aggrecan (313% of nonloaded values) but statistically significant decreases in collagenase mRNA levels (35% of nonloaded values). Cyclic tension also resulted in a statistically significant decrease in collagenase mRNA levels (66% of nonloaded values) and an increase in aggrecan mRNA levels (458% of nonloaded values) but no significant change in the mRNA levels for the other molecules. The results show that it is possible to alter mRNA levels for a subset of genes in scar tissue by supplying unique mechanical stimuli in vitro and thus that further investigation of scar engineering for potential reimplantation appears feasible.

Comparison of mRNA levels for matrix molecules in normal and disrupted human anterior cruciate ligaments using reverse transcription-polymerase chain reaction.

Midsubstance samples of anterior cruciate ligaments from seven normal human cadaver knees (16-50 years old) were harvested and compared with midsubstance pieces of scarred anterior cruciate ligaments from 30 patients (15-40 years old). RNA was isolated from each ligament, and the expression of type-I collagen, type-III collagen, biglycan, decorin, lumican, and tissue inhibitor of metalloproteinase-1 was evaluated by quantitative reverse transcription-polymerase chain reaction with use of beta-actin as the housekeeping gene. Data for injured ligaments were further compared statistically as a function of time after injury to better define patterns of cellular expression over time. Our hypothesis was that injured ligaments would show minimal cellular activity and decreasing activity over time. The results revealed that both normal and injured anterior cruciate ligaments contain cells that express mRNA for all molecules studied. However, cells in injured ligaments express much higher, but still proportional, quantities of message for type-I collagen and type-III collagen (p < 0.000001) and higher quantities of biglycan (p < 0.02) and tissue inhibitor of metalloproteinase-1 (p < 0.0003) than do cells in normal anterior cruciate ligaments. These levels remained elevated for longer than 1 year after injury. Linear regression analysis showed biglycan expression correlated with time from injury (r2 = -0.69; p = 0.007). These results collectively demonstrate that injured human anterior cruciate ligaments contain cells that express scar-like molecules and that the injured ligaments are likely continuing to remodel matrix over time. Furthermore, they suggest that human anterior cruciate ligaments have not failed to heal due to the failure of scar formation per se. The quality and quantity of this scar remain questionable; however, the possibility of its enhancement as a healing strategy for human anterior cruciate ligaments cannot be dismissed.

Decorin antisense gene therapy improves functional healing of early rabbit ligament scar with enhanced collagen fibrillogenesis in vivo.

Injured ligaments heal with scar tissue, which has mechanical properties inferior to those of normal ligament, potentially resulting in re-injury, joint instability, and subsequent degenerative arthritis. In ligament scars, normal large-diameter collagen fibrils have been shown to be replaced by a homogenous population of small collagen fibrils. Because collagen is a major tensile load-bearing matrix element and because the proteoglycan decorin is known to inhibit collagen fibrillogenesis, we hypothesized that the restoration of larger collagen fibrils in a rabbit ligament scar, by down-regulating the proteoglycan decorin, would improve the mechanical properties of scar. In contrast to sense and injection-treated controls, in vivo treatment of injured ligament by antisense decorin oligodeoxynucleotides led to an increased development of larger collagen fibrils in early scar and a significant improvement in both scar failure strength (83-85% improvement at 6 weeks; p < 0.01) and scar creep elongation (33-48% less irrecoverable creep; p < 0.03) under loading. This is the first report that in vivo manipulation of collagen fibrillogenesis improves tissue function during repair processes with gene therapy. These findings not only suggest the potential use of this type of approach to improve the healing of various soft tissues (skin, ligament, tendon, and so on) but also support the use of such methods to better understand specific structure-function relationships in scars.

In-vitro cyclic tensile loading of an immobilized and mobilized ligament autograft selectively inhibits mRNA levels for collagenase (MMP-1).

To test the hypothesis that loading conditions can be used to "engineer" ligament autograft behaviors, the effect of cyclic tension on the mRNA levels of matrix molecules and collagenase in in-vivo immobilized and mobilized 6-week rabbit medial collateral ligament (MCL) autografts was examined using an in-vitro system. Femur-[autograft MCL]-tibia complexes were subjected to a tensile stress of 4 MPa at 0.5 Hz for 1 min, followed by 14 min of rest. This 15-min testing cycle was repeated for 4 h. Semi-quantitative reverse transcrip-tase polymerase chain reaction (RT-PCR) was performed on RNA from mechanically treated MCL autografts, using rabbit-specific primer sets for types I and III collagen, biglycan, decorin, fibromodulin, lumican, versican, matrix metalloproteinase-1 (MMP-1, collagenase-1), MMP-13 (collagenase-3), and a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Interestingly, 4 h of culture of normal control MCLs led to increased mRNA levels for MMP-1 (P < 0.05), but there were no significant changes in MMP-13 mRNA levels. Total RNA levels in that normal MCL tissue were, however, decreased after culture (P < 0.05). In-vitro tensile loading of in-vivo mobilized autografts resulted in a significant increase in total RNA (185% of in-vitro non-loaded autografts). On the other hand, in-vitro tensile loading of in-vivo immobilized autografts resulted in no significant changes in total RNA levels compared with levels in non-loaded control grafts. MMP-1 mRNA levels in both the in-vivo mobilized (47% of non-loaded autograft) and in-vivo immobilized (38% of non-loaded autograft) MCL autografts were significantly lower than those in non-loaded control tissue following in-vitro tensile loading, but there were no significant changes in the mRNA levels for the seven other matrix molecules assessed. These results show that it is possible to selectively inhibit MMP-1 mRNA levels in autograft ligaments by supplying mechanical stimuli in vitro. The results also demonstrate that in-vivo immobilization leads to a decrease in the effects of subsequent in-vitro mechanical loading in such autografts with respect to total RNA levels. Collectively, these results demonstrate that both in-vivo and in-vitro loading have implications in the engineering of an ideal ligament graft.